A novel variant in the FLCN gene in a Chinese family with Birt–Hogg–Dubé syndrome

Abstract Background This study aimed to identify disease‐causing variants within a Chinese family affected by Birt–Hogg–Dubé syndrome (BHDS), which arises from an autosomal dominant inheritance pattern attributed to variants in the folliculin (FLCN) gene, recognized as a tumor suppressor gene. Methods A Chinese proband diagnosed with BHDS due to renal tumors underwent next‐generation sequencing (NGS), revealing a novel variant in the FLCN gene. Sanger sequencing was subsequently performed on blood samples obtained from family members to confirm the presence of this variant. Results A novel germline frameshift variant (NM_144997.5:c.977dup) was identified in five individuals among the screened family members, marking the first report of this variant. Additionally, a somatic frameshift variant (NM_144997.5:c.1252del) was detected in the renal tumors of the proband. No variant was detected in unaffected family members. Conclusions A novel heterozygous variant was identified in exon 9 of the FLCN gene, which broadens the spectrum of FLCN variants. We recommend that molecular analysis of the FLCN gene be performed in patients with suspected BHDS and their families.


| INTRODUCTION
Birt-Hogg-Dubé syndrome (BHDS) is a rare autosomal dominant disorder, initially proposed and named in 1977.It is caused by germline variants in the FLCN gene (Gene MIM number: 607273) and located on the short arm of chromosome 17p11.2(Birt et al., 1977).The penetrance of BHDS is generally high, although the disease's expression varies significantly among family members and across different families (Schmidt et al., 2005).Typical clinical manifestations include multiple pulmonary cysts (recurrent spontaneous pneumothorax), cutaneous fibrofolliculoma, and various histological types of renal tumors.Literature indicates that approximately 16%-30% of BHDS patients will develop renal tumors, typically around the age of 50, which is seven times higher than the general population.Renal tumor development usually occurs later than skin and lung symptoms (Hasumi et al., 2016).The disease exhibits wide phenotypic heterogeneity, and clear genotype-phenotype correlations have not yet been established (Furuya et al., 2020;Sattler et al., 2018).Notably, skin manifestations are often less prominent in Asian individuals, and pulmonary cysts may go unnoticed (Iwabuchi et al., 2018).Furthermore, due to insufficient awareness of the disease, physicians often treat each symptom individually, potentially overlooking the presence of BHDS, leading to a high rate of misdiagnosis and missed diagnoses.Thus, while BHDS is widely considered highly variable in its phenotype, this interpretation may slightly deviate from the actual clinical scenario.
In this study, we examined clinical and genetic data from a patient with BHDS-associated renal tumors in China and identified a novel variant in the FLCN gene region, which was confirmed by Sanger sequencing of her large family members.Our results provide genetic counseling for this family and enrich the FLCN gene mutation database, contributing to the exploration of the relationship between BHDS genotype and phenotype.

| Ethical compliance
The study was approved by the institutional review board of initiating center Jinling Hospital (ID Number: 2021NZKY-004-01).Written informed consent was obtained from all participants.

| Patient recruitment and evaluation
A 35-year-old female proband was diagnosed with a high suspicion of BHDS at Jinling Hospital.A retrospective analysis of her clinical and pathological data was conducted, and the family history of 21 relatives across four generations was collected.Written informed consent was obtained from all individuals for potential publication of identifiable images or data in this study.All subjects underwent skin screening by professional dermatologists.Computed tomography (CT) of the chest was performed to evaluate the pulmonary lesions, and abdominal ultrasound examinations were conducted to rule out renal involvement.

| Identification by next-generation sequencing
With their full notification and informed consent, peripheral blood and tumor samples from the proband were drawn and sent to a third-party company for sequencing.First, the DNA was fragmented, and the library prepared.Then, the entire coding regions including the intronic flanking sequences of FLCN were amplified by PCR.The software IDT2 was used to design primers for PCR.Sequences of PCR products were determined by the ABI 3100 Genetic Analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, United States).Finally, The DNA sequencing reaction of the proband was performed using the next-generation sequencing (NGS) method according to manufacturer's protocols.The multiple FLCN protein sequences were aligned using the program MUSCLE (version 3.6).The online databases, PolyPhen-2 (polymorphism phenotyping), and MutationTaster programs were used to predict the possible effects of variants on the function of the proteins.Please refer to the American College of Medical Genetics and Genomics (ACMG) for standards and guidelines on the interpretation of genetic variations.The GenBank reference sequence and version number for the FLCN transcript variant was NM_144997.5.The FLCN protein sequence and confirmed by use of the Mutalyzer program (http:// www.lovd.nl/ mutal yzer).

| Validation by Sanger sequencing
To validate the novel variants identified by NGS, 5 mL of peripheral blood was drawn from the proband's family members for Sanger sequencing to confirm the presence or absence of these variants.The sequencing products were purified and analyzed by Wuhan Yingjun Biotechnology Service Co., Ltd., and the results were interpreted using Chromas software.

| Clinical characteristics
In our study, the proband (III9) underwent robot-assisted laparoscopic partial nephrectomy surgery due to multiple left renal tumors.Surgery resulted in the complete resection of three tumors, ranging in diameter from approximately 1.5 cm to 3 cm (Figure 1a,b).Physical examination revealed multiple papules and brown freckles on her face and neck (Figure 1c,d), which are highly suggestive of BHDS fibrofolliculoma-like changes.However, she declined a further skin biopsy.Chest CT revealed bilateral lung cysts (Figure 1e), and she had been diagnosed with spontaneous pneumothorax at the age of 29.In her family, her grandfather (I1) died of gastric carcinoma, and her grandmother (I2) died of pancreatic adenocarcinoma without any symptoms of renal tumors.Seven family members, including the proband, developed BHDS-related symptoms.Specifically, her first uncle (II1) had multiple pulmonary cysts and renal tumors, leading to a radical nephrectomy.His daughter (III1) underwent partial nephrectomy for renal tumors several years later.The proband's mother (II10) and her third uncle (II5) both experienced spontaneous pneumothorax, and subsequent chest CT confirmed multiple pulmonary lesions.One aunt (II8) underwent radical nephrectomy due to renal tumor and also had lung cysts (Figure 1f), while her daughter (III7) was diagnosed with multiple lung cysts following pneumothorax.None of the affected patients, except for the proband, exhibited cutaneous lesions.None of her father's relatives presented any symptoms of BHDS.Patients who underwent surgery were followed up through outpatient or telephone visits and showed no signs of tumor metastasis or recurrence.The clinical characteristics of the living family members are summarized in Table 1.The pedigree of the family members included in the study is shown in Figure 2.

| FLCN gene variation analysis
A germline frameshift variant was identified in exon 9 of the FLCN gene (NM_144997.5:c.977dup) in the proband.The variant causes an alteration of alanine to glycine at position 327 of the coding protein, resulting in premature termination at position 63 and truncation of the polypeptide chain.Five out of 16 relatives were found to carry the same FLCN gene germline variant (II8;II10;III7;III9;IV5), indicating that this familial disorder is caused by the FLCN gene germline variant (Figure 4a,b).The FLCN variant (p.Ala327GlyfsTer63) has never been previously reported in ClinVar, LOVD, or VarCards genetic variation databases, nor in the 1000_CN, ESP6500, or other population databases.It is also not included in the ClinVar database and FLCN mutation database (http:// www.lovd.nl/ FLCN).According to ACMG guidelines, the variant was classified as potentially pathogenic.Additionally, a somatic frameshift variant (NM_144997.5:c.1252del) was also detected in the tumor cell DNA.This somatic variant is not cataloged in COSMIC v90 but is listed as pathogenic in LOVD.Notably, the tumor mutation burden (TMB) was 0.47 muts/Mb, and the microsatellite instability (MSI) status was microsatellite stable (MSS).No DNA copynumber abnormalities were found.The NGS statistics for the proband's FLCN gene are presented in Table 2.

| DISCUSSION
BHDS is a rare autosomal dominant genetic disease, primarily reported in White populations.The prevalence of its common features may vary based on researchers' specialty and interests, making the accurate prevalence remain unclear.Renal tumors are a relatively infrequent manifestation of BHDS and distinct from sporadic renal cell carcinoma (RCC).A comprehensive review of 204 families with at least one manifestation of BHDS indicated that individuals with the FLCN variant have an estimated 19% risk of developing renal tumors in males and 21% in females (Bruinsma et al., 2023).While BHDS has been reported in several Asian families, particularly in Japan and Korea, most patients exhibit primarily pulmonary manifestations (Furuya et al., 2016;Hu et al., 2021;Liu et al., 2022).Only about 3.6% of BHDS patients in Asia present with renal tumors, possibly due to insufficient knowledge about BHDS among Asian physicians and potential racial differences.In our study, among the eight variant positive or clinically positive family members, four individuals (50%) developed renal tumors.This incidence rate is higher than the reported range of 16%-30% (Hasumi et al., 2016).
The FLCN gene encodes the highly conserved tumor suppressor folliculin, which interacts with folliculininteracting protein 1 and 2 (FNIP1 and FNIP2), as well as AMP-activated protein kinase (AMPK), to regulate the mammalian target of rapamycin (mTOR) pathway.This pathway controls cell growth, proliferation, and survival.In a mice model (FLCN FLOX/FLOX/Ksp-Cre), specific knockout of FLCN in renal distal tubule and collecting duct cells leads to polycystic kidney disease and uremia, resulting in death within 3 weeks of birth (Wu et al., 2015).Alterations in FLCN within renal distal tubular cells promote tumorigenesis and the development of various histological tumor phenotypes.The mTOR pathway plays a crucial role in the growth of isogeneic transplant models (Wu et al., 2015) with FLCN alterations and human UOK257 xenograft models.Variants in the FLCN gene diminish the tumor suppressor effect of these signaling pathways, triggering renal tumors in renal distal tubules in accordance with the second-hit hypothesis (Vocke et al., 2005).Previous studies have reported the co-occurrence of somatic variants in the FLCN gene in both BHDS-associated renal tumors and parathyroid adenomas (Jha et al., 2023;Wang et al., 2022), which aligns with our findings and strongly supports this hypothesis (Daccord et al., 2020).
BHDS typically affects the skin, lungs, and kidneys.In rare cases, patients have also been reported to have colorectal cancer, salivary gland oncocytomas, melanomas, oral papules, and thyroid cancers, although it remains unclear whether FLCN variants are responsible for these manifestations (Daccord et al., 2020).Ethnicity appears to influence the presentation of BHDS.In North American and European patients, skin lesions are typically the predominant manifestation, followed by renal tumors at a later stage, with a low incidence of pneumothorax (Kluger et al., 2010).Conversely, BHDS patients in Asia often exhibit a "pulmonary phenotype," characterized by multiple pulmonary cysts and recurrent spontaneous pneumothorax, while cutaneous manifestations are rare and less typical (Furuya et al., 2016(Furuya et al., , 2020)).
The proband in our study exhibited a "renal-phenotype" of BHDS, characterized by multiple renal tumors, while pulmonary and cutaneous manifestations were relatively nonspecific.The International BHD Consortium recommends diagnostic FLCN gene testing for BHDS patients starting at age 20 (Berger et al., 2020).Despite numerous studies on genotype-phenotype correlations in BHDS patients, no significant associations have been found thus far (Sattler et al., 2018), suggesting that a larger cohort may be necessary to uncover meaningful correlations.
According to the Leiden Open Variation Database, over 280 unique variants have been identified throughout the coding region of the FLCN gene to date (Lim et al., 2010).A recognized variant hotspot involves the deletion or insertion of cytosine residues in exon 11, with nearly half of patients carrying either c.1285delC or c.1285dupC variants, consistent across European and Asian populations (Schmidt et al., 2005).Other patients exhibit intragenic deletions or duplications and pathogenic missense variants (Benhammou et al., 2011;Matsumoto et al., 2021).A Japanese study (Furuya et al., 2016) revealed two additional variant hotspots, c.1533_1536delGATG and c.1347_1353dupCCACCCT, in the Japanese BHDS population.In our study, we identified a novel frameshift variant (c.977dup) in exon 9 of the FLCN gene, which has not been previously reported in the literature or the FLCN mutation database (Lim et al., 2010;Zhou et al., 2022).
BHDS-associated renal tumors are typically bilateral and multifocal, characterized by low malignant potential but a lifetime risk of developing additional tumors (Daccord et al., 2020).It is important to differentiate it from other hereditary renal tumor syndromes, particularly tuberous sclerosis complex (TSC).BHDS-associated renal tumors exhibit diverse histological features, with HOCT being the most common (approximately 50%), followed by chRCC (30%) and clear cell renal cell carcinoma (9%).Eosinophilic chromophobe carcinoma and papillary renal cell carcinoma are rare occurrences (Pavlovich et al., 2002).
Preoperative diagnosis through imaging is challenging.For patients with HOCT, it is essential to conduct a detailed family history inquiry, perform chest CT to assess for pulmonary lesions, and carry out a thorough skin examination to determine the presence of BHDS.The 2015 diagnostic criteria for BHDS (Schmidt & Linehan, 2015) recommend the identification of FLCN gene germline variants through genetic testing as the optimal diagnostic standard.High-precision DNA sequencing analysis is particularly important, with a detection rate exceeding 90%.Currently, there are no specific treatments targeting the etiology of BHDS.Renal ultrasonography may not detect small or isoechoic HOCTs.Menko et al. (2009) recommend annual MRI scans for patients with BHDS starting at age 20 to screen for kidney cancer, although ultrasound can be used for preliminary screening, especially in institutions without access to MRI (Perdeaux & Solly, 2013).Most BHD-associated renal tumors rarely metastasize at the time of initial diagnosis, and partial nephrectomy remains the primary treatment to preserve renal function.Jikuya et al. (2022) found that the HIF-VEGF signaling pathway is not upregulated in the vascular system associated with BHD, indicating that angiogenesis inhibitors may not be effective treatments.The tumor of the proband showed strong expression of PD-L1, suggesting that immune checkpoint inhibitors may have potential benefits, consistent with previous research findings.Additionally, Jikuya et al. (2022) discovered high expression of the mesenchymal to epithelial transition factor (MET) in various histological types of BHDS-associated renal tumors, suggesting that MET inhibitors such as cabozantinib and crizotinib may provide promising therapeutic options for treating BHDSassociated renal tumors.
In conclusion, we identified a novel heterozygous frameshift variant (NM_144997.5:c.977dup) in exon 9 of the FLCN gene.This discovery broadens the spectrum of FLCN gene variants in the Chinese population.Related functional studies examining the consequences of the identified FLCN gene variants should be conducted next, which could provide deeper insights into the pathogenesis of BHDS.

F
I G U R E 1 Clinical characteristics of the proband and her aunt.(a) Computed tomography of the proband's abdomen.The red arrow indicates multiple tumors in the left kidney.(b) Three renal tumors of the proband, ranging in diameter from 1.5 to 3 cm, after partial nephrectomy.(c) Multiple fibrous papules on the proband's face.(d) Multiple fibrous papules on the proband's neck.(e) Computed tomography of the proband's chest indicated multiple pulmonary bullae on the left side.(f) Chest computed tomography image of the proband's aunt showed multiple pulmonary bullae.
Clinical information and genotypes of individuals from the Chinese family with BHDS.

F
Pathological staining images of renal tumors in the proband (×200).(a) Hematoxylin-eosin staining of tumors.Immunohistochemical analyses for Transcription Factor Binding To IGHM Enhancer 3 (TFE3) (b), Transcription factor EB (TFEB) (c).F I G U R E 4 Sequence analysis of the FLCN gene.(a) The chromatograms show a novel heterozygous frameshift variation (red arrow) of c.977dup in the proband; (b) Sequencing result of a normal sequence in the unaffected son.
T A B L E 2